Journal: EMBO Molecular Medicine

Article Title: B cell lineage reconstitution underlies CAR-T cell therapeutic efficacy in patients with refractory myasthenia gravis

doi: 10.1038/s44321-024-00043-z

Figure Lengend Snippet: ( A ) A schematic overview of the time points at which patients receive different treatments before CAR-T infusion. # Patient MG-1 was treated with IVIG 2 g/Kg + intravenous pulse steroid 500 mg* 3days for myasthenia crisis. ( B ) A schematic overview of CAR-T treatment procedure. CAR T-cell kinetics are shown by the CAR copies per μg genomic DNA at serial time points post infusion detected by droplet digital PCR. ( C ) Representative plots showing FACS analysis stained for CAR-T cells with FITC-labeled human BCMA Fc tag protein and APC/Cy7 anti-human CD3 antibody in patient MG-1 at day 10 after CAR T-cell infusion. CAR T-cell percentage in circulating CD3 + T lymphocytes at serial time points after treatment. ( D ) Timelines of patients with cytopenia of grade 3 or higher at baseline and indicated time points after CAR T-cell infusion. BL baseline. Kinetic changes in numbers of circulating total white blood cells, neutrophils, monocytes and platelets. ( E ) Heatmap depicting protein levels of inflammatory mediators in blood following CAR T-cell infusion. Interleukin IL, TNF tumor necrosis factor, IFN interferon, CRP C-reactive protein, PCT procalcitonin. Average levels are normalized from the baseline. .

Article Snippet: Website links for the antibodies used in the flow cytometry are following: PerCP/Cyanine5.5 anti-human CD45 Antibody (Biolegend, 304028): https://www.biolegend.com/en-us/products/percp-cyanine5-5-anti-human-cd45-antibody-4240 ; FITC anti-human CD3 antibody (BD Biosciences, 561802): https://www.bdbiosciences.com/zh-cn/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/fitc-mouse-anti-human-cd3.561802 ; PE/Cyanine7 anti-Human CD4 (BD Biosciences, 560649): https://www.bdbiosciences.com/zh-cn/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/pe-cy-7-mouse-anti-human-cd4.560649 ; APC/Cyanine7 anti-Human CD8 (BD Biosciences, 557834): https://www.bdbiosciences.com/zh-cn/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/apc-cy-7-mouse-anti-human-cd8.557834 ; APC anti-human CD19 Antibody (Biolegend, 302212): https://www.biolegend.com/en-us/products/apc-anti-human-cd19-antibody-715 ; PE anti-human CD16 Antibody (Biolegend, 302056): https://www.biolegend.com/en-us/products/pe-anti-human-cd16-antibody-569 ; PE anti-human CD56 Antibody (Biolegend, 318306): https://www.biolegend.com/en-us/products/pe-anti-human-cd56-ncam-antibody-3796 ; FITC anti-Human CD38 (BD Biosciences, 567147): https://www.bdbiosciences.com/en-us/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/fitc-mouse-anti-human-cd38.567147 ; PerCP/Cyanine5.5 anti-Human CD27 (BD Biosciences, 560612): https://www.bdbiosciences.com/zh-cn/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/percp-cy-5-5-mouse-anti-human-cd27.560612 ; PerCP anti-Human CD45 (BD Biosciences, 347464): https://www.bdbiosciences.com/zh-cn/products/reagents/flow-cytometry-reagents/clinical-discovery-research/single-color-antibodies-ruo-gmp/percp-mouse-anti-human-cd45.347464 ; APC/Cyanine7 anti-human CD3 Antibody (Biolegend, 344818): https://www.biolegend.com/en-us/products/apc-cyanine7-anti-human-cd3-antibody-6940 ; FITC-labeled human BCMA Fc tag protein (Acrobiosystems, BCA-HF254): https://www.acrobiosystems.cn/P875-FITC-Labeled_Human_BCMA_%7C_TNFRSF17_Protein_Fc_Tag.html .

Techniques: Digital PCR, Staining, Labeling

Journal: The Journal of Clinical Investigation

Article Title: B cell maturation antigen–specific CAR T cells are clinically active in multiple myeloma

doi: 10.1172/JCI126397

Figure Lengend Snippet: (A) Baseline peripheral blood serum concentration of sBCMA and APRIL for subjects (sub) were significantly increased and decreased, respectively, compared with a panel of healthy donors (HD, n = 6) (P = 0.017, and P < 0.001, respectively, Mann-Whitney). Baseline BAFF concentrations were not significantly different. Median concentrations are depicted by red lines. (B) Serial sBCMA concentrations decline after CART-BCMA infusions more significantly in hematologic responders (PR/VGPR/CR/sCR) than in nonresponders (MR/SD/PD) before day 28 (P < 0.001). After day 28 the slopes of the curves are not significantly different between groups (P = 0.429). The estimation was based on a linear random intercept mixed effects model on log10-transform sBCMA that included 2 piecewise linear splines connected at day 28; P values were determined based on z test for the regression coefficient of interest or a linear combination of the coefficients. Mean concentration (ng/ml) + SEM are depicted. (C) Representative examples of BCMA expression on MM cells by flow cytometry. See Supplemental Figure 9 for gating strategy. FMO, fluorescence minus one. (D) BCMA mean fluorescence intensity (MFI) on MM cells over time in 18 subjects with evaluable serial bone marrow aspirates. Median MFI was significantly different between pretreatment (pre-tx) and day 28 (D28) for responders (4000 vs. 944, P = 0.02, paired t test) but not for nonresponders (2704 vs. 2140, P = 0.19). Median MFI was not significantly different between pre-tx and day 90 (D90) for responders (4000 vs. 2022, P = 0.26). *Subject 15 had no detectable MM cells at D28. #Subject 03 had no detectable MM cells at D45 (D28 not done) and too few MM cells to characterize at D90. D164 marrow is depicted at D90 time point.

Article Snippet: Measurement of soluble BCMA, BAFF, and APRIL concentrations in serum was performed by ELISA using antibody sets for human BCMA (DY193), APRIL (DY884B), and BAFF (DT124-05) from R&D Systems.

Techniques: Concentration Assay, MANN-WHITNEY, Expressing, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: Tailored strategies for improved control of CAR-T cells in multiple myeloma

doi: 10.3389/fimmu.2026.1740345

Figure Lengend Snippet: Efficiency of ADCC and ADC on SLAMF7 and CD19 CAR-T cells in vitro ; (A) Schematic depiction of EGFR-specific ADCC; (B, C) CD19 and SLAMF7 CAR-T cells (CD4:CD8 1:1) were incubated with EGFR antibody cetuximab [50 µg/ml] and cocultured with effector cells (CFSE stained, PBMC w./w.o. NK cells, E:T 50:1 and NK cells, E:T 10:1) for 24 h, CAR-T cell elimination through ADCC is dependent on NK cell availability and CAR-T cell construct, Representative Flow cytometry plots (B) and statistical analysis ((C) n=3) are shown; (D) Lymphocyte (n=12) and NK cell (n=8) counts are reduced after lymphodepleting chemotherapy (d-5, -4 and -3) and only gradually recover; (E) Schematic depiction of the ADC assay; (F) BCMA expression of targeted cells via flow cytometry, (G, H) Cells expressing BCMA or not, were incubated with BCMA targeting ADC belantamab-mafodotin [50 µg/ml]; SLAMF7 CAR-T cells, UTD ctrl T cells (72 h; (G) ) and the human MM cell line OPM-2 (48 h & 72 h; (H) ) are shown; Control antibody: Human IgG1 Isotype Control; n=3 independent donors; Each experiment was performed in triplicates; Two-way ANOVA statistic test was performed using GraphPad Prism 9; Abbreviations: ADC, Antibody-drug conjugate; ADCC, Antibody-dependent cell cytotoxicity; ALC, Absolute Lymphocyte Count; CAR, Chimeric antigen receptor; Ctrl, Control; MM, Multiple myeloma; SLAMF7, SLAM Family member 7 (CD319); UTD, Untransduced. **** = P ≤ 0,0001; *** = P ≤ 0,001; * = P ≤ 0,05.

Article Snippet: Antibodies used in this research report were specific for BCMA-Antibodies used in this research report were specific for BCMA (Miltenyi Biotec; 130-119-152), EGFR (Cetuximab, in-house labelled), CD8 (Miltenyi Biotec; 130-110-683) and CD4 (Miltenyi Biotec; 130-114-534).

Techniques: In Vitro, Incubation, Staining, Construct, Flow Cytometry, Expressing, Control

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: Schematic of BCMA-Nb radiolabeling with (A) [ 18 F]FPy and (B) [ 131 I]I.

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Radioactivity

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Kinetic binding assay of BCMA nanobody to human BCMA protein measured by biolayer interferometry using 100nM anti-BCMA-nanobody (B) Representative flow cytometry histograms of BCMA expression in MC38-BCMA and MC38-WT cells (C) Immunohistochemistry and Immunofluorescent staining of BCMA in excised tumors (D) Saturation binding assays of [ 18 F]FPy-BCMA Nb in MC38-BCMA cells (E) Representative PET/CT coronal images of MC38-BCMA and MC38-WT tumor model (150-250 mm 3 ), injected with 3.7 MBq [ 18 F]FPy-BCMA-Nb at 2 h post-injection. PET scans are normalized to the same scale. Data are presented as mean ± SD (n = 6 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Binding Assay, Flow Cytometry, Expressing, Immunohistochemistry, Staining, Positron Emission Tomography-Computed Tomography, Injection

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Flow cytometry analysis of BCMA expression in H929 and RPMI8226 cell lines. (B) Immunofluorescent staining of H929 cells using anti-BCMA antibody (green) and DAPI (blue). Saturation binding assay to assess the specific binding and dissociation constant of [ 18 F]FPy-BCMA-Nb in (C) H929 and (D) RPMI8226 cells. (E) Internalization of [ 18 F]FPy-BCMA-Nb in H929 cells over 180 minutes. (F) 3 h post-injection uptake of [ 18 F]FPy-BCMA-Nb in the femur/knee region and (G) spine of the human H929 and RPMI8226 MM model compared to healthy mice and blocking. White arrows indicate tumor cells in the bone marrow. Relative quantitative uptake of the tracer in the H929 human MM model with higher BCMA expression in spine, femur, arm, skull bone marrow, and kidney under (H) pre-blocking and (I) post-blocking conditions . Data are presented as mean ± SD (n = 6 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Flow Cytometry, Expressing, Staining, Saturation Assay, Binding Assay, Injection, Blocking Assay

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Schematic illustration of the timeline, treatment regimen and efficacy study for the radiotherapy study. (B) Representative BLI images of mice pre- and post-treatment regimen and (C) overall survival percentage across all groups, including Control (PBS), 7.4 MBq and 18.5 MBq of [ 131 I]I-BCMA-Nb, compared to 18.5 MBq of [¹³¹I]I-non-specific-Nb treated cohorts, as determined by Kaplan-Meier analysis. Data are presented as mean ± SD (n = 8 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Control

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Representative Sagittal [ 18 F]FDG PET images of healthy mice compared to treatment groups (PBS, 7.4 MBq and 18.5 MBq of [ 131 I]I-BCMA-Nb,) acquired pre- and post-treatment regimen. Quantitative analysis of [ 18 F]FDG uptake in bone marrow of the (B) spine, (C)femur, (D) arm, and (E) skull across all groups pre-, during and post-treatment. Gray areas are defined as baseline based on [ 18 F]FDG of healthy mice. Data are presented as mean ± SD (n = 8 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques:

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: Changes in (A) body weight during the treatment regimen. (B) Complete cell blood counts for mice pre- and post-treatment in all groups including PBS, injected with 7.4 MBq and 18.5 MBq of [ 131 I]I-BCMA-Nb and 18.5 MBq of [ 131 I]I-non-specific-Nb. Orange-shaded cells indicate values significantly different from standard NSG mouse ranges (p<0.05). Data are presented as mean ± SD (n = 8 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Injection

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Immunohistochemistry staining and (B) quantitative analysis of BCMA, (C) immunofluorescence staining for and (D) quantitative analysis of Ki67 proliferation marker in femur and spine bone marrow sections of H929 human MM-bearing mice, before and after treatment, compared with healthy mice.

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Marker